Abstract
As one of the main energy metabolism organs, kidney has been proved to have high energy requirements and are more inclined to fatty acid metabolism as the main energy source. Long-chain acyl-CoA dehydrogenases (LCAD) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD), key enzymes involved in fatty acid oxidation, has been identified as the substrate of acetyltransferase GCN5L1 and deacetylase Sirt3. Acetylation levels of LCAD and beta-HAD regulate its enzymes activity and thus affect fatty acid oxidation rate. Moreover, immunoprecipitation is a key assay for the detection of LCAD and beta-HAD acetylation levels. Here we describe a protocol of immunoprecipitation of acetyl-lysine and western blotting of LCAD and beta-HAD in palmitic acid treated HK-2 cells (human renal tubular epithelial cells). The scheme provides the readers with clear steps so that this method could be applied to detect the acetylation level of various proteins.
Keywords: Immunoprecipitation, Acetylation, LCAD, Beta-HAD, Palmitic acid (PA), HK-2 cells
Background
Post-translational modifications (PTMs) enable the cell with a highly dynamic mechanism for regulation of cellular pathways (Zhao et al., 2010). Acetylation has emerged as one of the major post-translational protein modifications. Accumulating evidences indicate that acetylation rivals phosphorylation as a regulatory modification in mitochondria (Henriksen et al., 2012). Over 60% of mitochondrial proteins are acetylated, being of notice, which were involved in energy metabolism such as tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), fatty acid oxidation and amino acid metabolism (Hirschey et al., 2010; Hebert et al., 2013). Acetylation is also considered to be one of the main adaptive mechanisms for mitochondria in sensing nutritional challenges. Under the condition of excess energy supplement, mitochondrial proteins are hyperacetylated. Insufficient energy availability leads to a decreased acetylation level of mitochondrial proteins (Schwer et al., 2009). A large number of mitochondrial metabolic enzymes are acetylated in diabetes, including the FAO enzymes LCAD and beta-HAD (Kosanam, 2014). Dharendra et al. indicated that the upregulation of acetylation status of mitochondrial fatty acid oxidation enzymes promoted fatty acid oxidation in the heart of high fat diet mice (Thapa et al., 2018). Our previous study also found that the acetylation levels of FAO enzymes LCAD and β-HAD were enhanced in HK-2 cells treated with palmitic acid (PA) (Lv et al., 2019). Protein lysine acetylation refers to covalent addition of acetyl group to lysine residue (N-lysine acetylation). At present, since there are no acetylated antibodies of LCAD and HAD, the detection of LCAD and HAD acetylation levels mainly depends on immunoprecipitation analysis which mainly concentrates the acetylated proteins by using acetyl-lysine antibody and then detects the content of acetylated LCAD and acetylated HAD by using LCAD antibody and HAD antibody respectively (Figure 1).Figure 1. Flowchart of IP
Materials and Reagents
Equipment
Procedure
Data analysis
The acetylation level of LCAD or beta-HAD was presented by ratio of acetylated LCAD to total LCAD or acetylated HAD to total HAD.
Recipes
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Grants No. 81770729 and 91749111) and Shandong Province Taishan Scholar Project (Grants No. tsqn 20161073). This protocol was adapted from our previous work (Lv et al., 2019).
Competing interests
The authors declare that they have no conflict of interest.
References
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